Metalloenzymatic reactions

To understand all details of metalloenzymatic reactions, enormous efforts, both experimental and theoretical, have been exerted over the last decades. While experiments (e.g., X-ray crystallography, various spectroscopic techniques, electrochemistry) are crucial in initial phases of our understanding of a particular system, theoretical calculations complement these data by providing a unique one-to-one structure-energy mapping. Our efforts in the past years focused on two coupled binuclear systems: tyrosinase with a dicopper catalytic core,^3-7 detailed below, and non-heme diiron (NHFe₂) Δ⁹-desaturase.^1,2

Reaction mechanism of tyrosinase (Ty), a metalloenzyme featuring coupled binuclear copper active site

Tyrosinase (Ty) is a ubiquitous oxidase, involved in two distinct reactions: (1) conversion of L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA) and (2) subsequent oxidation of L-DOPA to L-DOPAquinone. These elementary reactions are the initial and rate-limiting steps (RLS) in the synthesis of melanins. Melanins are omnipresent photoprotective biopolymer pigments, with major implications for essential biological processes, ranging from fruit browning to severe human diseases, such as skin cancer. Ty is also involved in Parkinson’s disease, because of its ability to oxidize dopamine to form melanin in the brain. Due to the key catalytic role of tyrosinase in melanogenesis, it is an important molecular and therapeutic target.

We explored the full catalytic cycle of Ty, starting from oxy-Ty (Ty + O₂).⁴ By performing 500 ns MD simulation and extensive QM/MM modeling we elucidated the hydration level of the [Cu₂O₂] active site of oxy-Ty. It enabled us to find its best possible structural description, which was also confirmed by correlation of computed data with experimental resonance Raman spectra.

In the next step, by correlation of exhaustive QM/MM and QM-cluster modeling with kinetic and spectroscopic experiments, we clearly identified and characterized the elusive ternary catalytic intermediate (Ty+O₂+substrate).⁵ This allowed us to explore the monooxygenation with methyl 4-hydroxybenzoate, and to validate the revealed mechanism with different para-substituent monophenols, showing an interesting reversal in the RLS of the reaction.⁶

Ongoing efforts are focused on catechol oxidation by Ty which is the final step in the full Ty catalytic cycle. In the future, we will study the other members of the coupled binuclear copper (CBC) family - catechol oxidase and o-aminophenol oxidase, to understand the varying chemoselectivity across the CBC family.⁷

References:

1. Bím, D.; Chalupský, J.; Culka, M.; Solomon, E. I.; Rulíšek, L.; Srnec, M.: Proton-Electron Transfer to the Active Site Is Essential for the Reaction Mechanism of Soluble Δ9-Desaturase. J. Am. Chem. Soc. 2020, 142, 10412−10423. https://doi.org/10.1021/jacs.0c01786

2. Tupec, M.; Culka, M.; Machara, A.; Macháček, S.; Bím, D.; Svatoš, A.; Rulíšek, L.; Pichová, I.: Understanding desaturation/hydroxylation activity of castor stearoyl Δ9-desaturase through rational mutagenesis. Comput. Struct. Biotechnol. J. 2022, 20, 1378–1388. https://doi.org/10.1016/j.csbj.2022.03.010

3. Stańczak, A.; Chalupský, J.; Rulíšek, L.; Straka, M.: Comprehensive Theoretical View of the [Cu2O2] Side-on-Peroxo-/Bis-μ-Oxo Equilibria. ChemPhysChem 2022, 23, e202200076. https://doi.org/10.1002/cphc.202200076

4. Kipouros, I.; Stańczak, A.; Culka, M.; Andris, E.; Machonkin, T. R.; Rulíšek, L.; Solomon. E. I.: Evidence for H-bonding interactions to the m-h2:h2-peroxide of oxy-tyrosinase that activate its coupled binuclear copper site. Chem. Commun. 2022, 58, 3913‑3916. https://doi.org/10.1039/D2CC00750A

5. Kipouros, I.; Stańczak, A.; Ginsbach, J. W.; Andrikopoulos, P. C.; Rulíšek, L.; Solomon, E. I.: Elucidation of the tyrosinase/O2/monophenol ternary intermediate that dictates the monooxygenation mechanism in melanin biosynthesis. Proc. Natl. Acad. Sci. 2022, 119, e2205619119. https://doi.org/10.1073/pnas.2205619119

6. Kipouros, B. I.; Stańczak, A.; Dunietz, E. M.; Ginsbach, J. W.; Srnec, M.; Rulíšek, L.; Solomon, E. I. Experimental Evidence and Mechanistic Description of the Phenolic H-Transfer to the Cu₂O₂ Active Site of oxy-Tyrosinase. J. Am. Chem. Soc. 2023, 145 (38), 22866–22870. https://doi.org/10.1021/jacs.3c07450

7. Stańczak, A.; Kipouros, I.; Eminger, P.; Dunietz, E. M.; Solomon, E. I.; Rulíšek, L., Coupled Binuclear Copper Sites in Biology: An Experimentally-Calibrated Computational Perspective. Coord. Chem. Rev. 2025, 525, 216301. https://doi.org/10.1016/j.ccr.2024.216301

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Metalloenzymatic reactions